Design of 99mTc-DTPA-CLP and Preliminary Evaluationin Rats

Burcu Altıparmak, Fatma Y. Lambrecht* andOzge Er

Department of Nuclear Applications, Institute of NuclearScience, Ege University, Bornova, Izmir 35100, Turkey*Corresponding author: Fatma Yurt Lambrecht,fatma.yurt.lambrecht@ege.edu.tr

Radiopharmaceuticals are localized in (malignant)tumor tissues by different mechanisms. One of thesemechanisms, gelatinase enzyme activity, is associatedwith poor prognosis in cancer patients and potentialtargets for tumor imaging. There are some gelatinasesto be associated with metastatic potential for tumorimaging to possibly predict metastases. In this study,a cyclic decapeptide conjugate, DTPA-CLP(DTPA-Cys-Leu-Pro-Gly-His-Trp-Gly-Phe-Pro-Ser-Cys),was selected as a peptide conjugate because of itsselective inhibitory activity toward gelatinases.Peptide-conjugated DTPA-CLP was labeled with 99mTcwith a radiolabeling efficiency of 97.0 � 2.8%. Afterdetermining optimization conditions for radiolabeling, abiodistribution study of radiolabeled peptide in albinoWistar rats was performed. According to biodistribu-tion data, 99mTc-DTPA-CLP showed high uptake in thelung, liver, uterus, and spleen. These results show that99mTc-DTPA-CLP may be used for the imaging of gela-tinase activity in metastatic tumors.

Key words: DTPA-CLP, gelatinase, HWGF-containing peptideconjugate, matrix metalloproteinases, radiolabeled peptide

Received 18 July 2013, revised 12 September 2013 andaccepted for publication 15 October 2013

Matrix metalloproteinases (MMPs) comprise a family ofenzymes that degrade the basement membrane andextracellular matrix. Matrix metalloproteinases play majorroles in tissue remodeling and cell migration during mor-phogenesis and wound healing. Abnormal expression ofMMPs can occur in cancer, possibly enabling tumor cellsto attack the extracellular matrix and generate metastasesat sites distant from the primary tumor. Indeed, high MMPlevels are associated with poor prognosis in cancerpatients (1). Matrix metalloproteinases, a family ofZn2+-dependent endopeptidases, contain particularlyMMP-2 (gelatinase A, 72-kDa type IV collagenase) andMMP-9 (gelatinase B, 92-kDa type IV collagenase) (2).

The overproduction of MMPs has been linked tomalignancy in a variety of tumors, including brain, colon,lung, bladder, melanoma, and breast. A negativeassociation between increased MMP expression andprognosis has been well documented with the meta-static phenotype of carcinomas, especially breast cancer(3,4). A large number of MMP inhibitors have beendeveloped as therapeutic agents, some of which arecurrently in clinical trials. Among MMP inhibitors, weselected a cyclic decapeptide as the mother compoundbecause of the clinical and radiopharmaceutical utility ofpeptide-based radiopharmaceuticals. MMPs are generallyinhibited by cyclic decapeptides containing the aminoacid sequence histidine-tryptophan-glycine-phenylalanine(HWGF) (3,5). Selective peptide inhibitors of MMP-2 andMMP-9 have been successfully labeled with radioiso-topes, such as 68Ga, 111In, 99mTc, 125I, 64Cu, evaluatedas radiotracers for imaging gelatinase activity in meta-static tumors (2,5–7). For instance, DOTA-CTT conjugatewas labeled with 64Cu (half-life 12.70 h), and its in vitro

and in vivo results show that 64Cu-DOTA-CTT had lowtumor uptake due to poor in vivo stability of the complex.For this reason, it was suggested that 64Cu-DOTA-CTTis not an optimal PET imaging agent (3). On the otherhand, the bioevolution of the 111In-DTPA-CTT (111In half-life: 2.83 d) was carried out in normal mice andtumor-bearing mice in the study by Hanako et al. Theyfound that 111In-DTPA-CTT has low levels of radioactivityin the liver and kidney in normal mice because of theincrease in hydrophilicity and its negative charge. Hence, itwas suggested that the data of tumor-bearing micesupport the validity of the chemical design of 111In-DTPA-CTT for reducing accumulation in non-target tissues(2).

In the current study, DTPA-CLP (DTPA-Cys-Leu-Pro-Gly-His-Trp-Gly-Phe-Pro-Ser-Cys), a cyclic decapeptideconjugate, contains the HWGF sequence and is selecteddue to its demonstrated selective inhibitory activity of gela-tinases. Due to this property, the peptide was preferredand firstly it was labeled with 99mTc. As known already,99mTc is the most commonly used medical radioisotopebecause of some advantages: short half-life (6 h), gammaemitter (140 keV gamma rays), generator product, etc.After the optimization of radiolabeling conditions of thepeptide conjugate, biodistribution study was performed innormal albino Wistar rats.

362 ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12253

Chem Biol Drug Des 2014; 83: 362–366

Research Article

Methods and Materials

Radiochemical synthesis of 99mTc-DTPA-CLPOne milligram of DTPA-CLP was dissolved in 20 mL ofwater and kept on a cold block. DTPA-CLP solution(20 lL, pH 7) was transferred to a vial, and SnCl2 2H2Oaq. solution (25 lg/25 lL) was added and then mixedwell. After 15-min incubation, fresh 99mTc (2-3 mCi) wasadded into the vial and left at room temperature for10 min. The labeling efficiency of 99mTc-DTPA-CLP wasdetermined by radio thin-layer chromatography (RTLC),using ITLC-SG plates and radio high-performance liquidchromatography (RHPLC). In RTLC method, two differentsolvent systems [0.9% sodium chloride solution and ACD(citrate–dextrose buffer solution (Eczacıbas�ı-Baxter, Tur-key)] were used as mobile phases, and the strips werescanned on a TLC scanner (Bioscan AR-2000, Washing-ton DC, USA). For the RHPLC analysis, we used theprocedures described by Kuhnast et al. (2004). A low-pressure gradient HPLC system (Shimadzu, Japan) wasused with LC-10Atvp quaternary pump and UV detector(Shimadzu SPD-10Atvp, Macherey-Nagel, EC 250/4, 6Nucleodur 100-5 C18 column) and equipped with a Cd(Te) RAD-501 single channel analyzer. The HPLC processwas using 0.1% TFA/acetonitrile (90%) and 0.1% TFA/water (10%) at a flow rate of 1 mL/min. The UV detectorwas settled at 220 nm. The radiochemical purity of radio-labeled conjugate was checked also using paper electro-phoresis method. Paper electrophoresis procedure wascarried out in a Gelman electrophoresis chamber. Cath-ode and anode poles and application points on celluloseacetate strips were marked and moistened with 0.9%NaCl solution. The radiolabeled peptide conjugate wasset on one strip and placed in the chamber. Standingtime and applied voltage were set at 90 min and 250V,respectively. And then, the strips were scanned using theTLC scanner.

The effect of parameters on radiolabeling yield of99mTc-DTPA-CLP

Effect of reaction pH on labeling yieldTo investigate the effect of reaction pH on labeling yield,DTPA-CLP peptide was labeled with 99mTc at pH 1, 3, 5,and 7. pH of DTPA-CLP peptide (10 lg/200 lL) wasadjusted, and 25 lL SnCl2 (1 mg/1 mL) solution wasadded. After 15-min incubation, fresh 99mTc (2-3 mCi) wasadded to the vial for 10-min incubation at room tempera-ture. The quality control was performed using RTLCmethod.

Effect of amount of tin chloride on DTPA-CLPlabeling yieldDTPA-CLP peptide was labeled 99mTc using 6.25, 12.5,25 37.5, and 50 lg SnCl2. DTPA-CLP peptide was kept

at pH 7, and SnCl2 solution was added into the tube. Afterincubation for 15 min, labeling was performed by addingfresh 99mTc (2–3 mCi). The mixture was incubated at roomtemperature for 10 min. Quality control was performedusing RTLC method.

Stability of 99mTc-DTPA-CLP in human serumTwo hundred microliters of the labeled peptide conjugatewas mixed with 500 lL of human serum. The mixture wasincubated at 370 C and analyzed in fixed time (30, 60,120, 180, and 1440 min) using RTLC method.

LipophilicityThe lipophilicity (logP) of the radiolabeled conjugate wasassessed as follows: the radiolabeled conjugate was pre-pared under optimum conditions (pH 7, 25 lg SnCl2),and 99mTc-DTPA-CLP (100 lL) was added to a premixedsuspension of 3 mL n-octanol in 3 mL water. The result-ing solution was mixed for an hour at room temperatureand allowed to stand for the formation of phases. Onehundred microliters of each phase was removed andcounted using a Cd(Te) RAD-501 single-channel ana-lyzer.

Biodistribution studyThe animal experiment was approved by the AnimalEthics Committee of Ege University and performed inaccordance with the published guidelines. The biodistri-bution of the radiolabeled conjugate in organs was inves-tigated using albino Wistar rats of weight 100–150 g.99mTc-DTPA-CLP (specific activity 13,88 Bq/nmol) wasintravenously injected to the rats. The rats were killed atfixed time (30, 60, and 120 min) after postinjection, andsome organs were removed, weighed, and countedusing Cd(Te)-RAD-501 single-channel analyzer. Theorgans’ activities were expressed as percent of radioac-tivity per gram of organ (%ID/g). Three rats were usedfor each fixed time.

Statistical analysisStatistical differences in the mean values of measuredactivities were evaluated by univariate variance andPearson’s correlation analyses using SPSS 10 software(IBM, Chicago, IL, USA). Probability values < 0.05 wereconsidered to be significant.

MaterialsPeptide DTPA-CLP (DTPA-Cys-Leu-Pro-Gly-His-Trp-Gly-Phe-Pro-Ser-Cys) was purchased from PiChem Inc.(Graz, Austria). Other reagents were purchased fromSigma-Aldrich Chemical Co. (Munich, Germany).Na99mTcO4 was supplied by the Department of Nuclear

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Labeling DTPA-CLP with 99mTc and Evaluation in Rats

Medicine, S� ifa University, as 99Mo/99mTc generator eluent(Monrol, Inc., Istanbul, Turkey).

Results and Discussion

Quality control of 99mTc-DTPA-CLP: DTPA-CLP (10 lg/200 lL) was labeled with 99mTc at pH 7 using 25 lg ofstannous chloride dehydrate as a reductant. The radio-chemical purity of the product was evaluated by RTLC,which successfully resolved labeled conjugate from R/H99mTc and free 99mTc. The Rf values of 99mTc-DTPA-CLP,free 99mTc, and R/H 99mTc were 0.03, 0.91, and 0.02,respectively, when using 0.9% NaCl as the developingsolution. When ACD was used as the developing solution,observed Rf (Relative front) values were 0.04, 0.92, and0.81, respectively. In addition, Rt (retention time) valuesobtained from RHPLC were 2.35 and 4.70 min for99mTc-DTPA-CLP and free 99mTc, respectively. The label-ing efficiency of 99mTc-DTPA-CLP was also evaluatedusing a paper electrophoresis method. The results showedthat 99mTc-DTPA-CLP had a negative charge. The dataobtained by the chromatograms indicated that the radiola-beling efficiency of 99mTc-labeled DTPA-CLP was97.0 � 2.8%. To determine the optimal pH for labelingefficiency, pH of the reaction mixture was varied from 3 to7 while all other experiment conditions remained constant(10 lg/200 lL peptide and 25 lg stannous chloride dehy-drate). The effect of pH on labeling efficiency is illustratedin Figure 1. At pH 1, 3, 5, and 7, the yield of 99mTc-DTPA-CLP was 93.6, 95.9, 97.7, and 98.3%, respectively. It isshown that labeling yields of 99mTc-DTPA-CLP are veryhigh at each pH. Similarly, the optimal amount of stannouschloride dehydrate was assayed by varying the amountbetween 6.25 and 50 lg while keeping the pH 7 andamount of DTPA-CLP constant.

The effect of concentrations of stannous chloride dehy-drate is indicated in Figure 2. At 6.25, 12.5, 25, 37.5, and50 lg concentrations of stannous chloride, the yield of99mTc-DTPA-CLP was determined as 62.1, 76.5, 97.7,92.9, and 68.9%, respectively. The optimum conditions of99mTc-DTPA-CLP are fixed as pH 7; peptide concentration

10 lg/200 lL; and the amount of stannous chloride25 lg.

99mTc-DTPA-CLP was quite stable in vitro up to 2 h. Thepercentage of labeled conjugate was also 83.1% at 24 h.In serum, radiolabeled conjugate was fairly stable for up to24 h (Figure 3), indicating that 99mTc-DTPA-CLP may besuitable for nuclear imaging. In the study conducted bySprague et al., 64Cu-DOTA-CTT and 64Cu-DOTA-STTconjugates were investigated. 64Cu-DOTA-CTT conjugatewas stable for up to 6 h, but stability decreased toapproximately 41.7 � 8.1% after 24 h (3). Kuhnast et al.

(5) found that 125I-radiolabeled cyclic HWGF decapeptidewas stable up to 1 h in serum. Lipophilicity analysis indi-cated that the experimental logP value of 99mTc-DTPA-CLP is 0.12 � 0.0. The lipophilicity of 125I-radiolabeledcyclic HWGF decapeptide was low (log P = 0.36) in astudy by Kuhnast et al. (5). Moreover, it has been demon-strated that 111In-DTPA-CTT is hydrophilic and possessesa negative charge (2). Our results are quite similar totheirs.

Biodistribution resultsThe biodistribution results of 99mTc-DTPA-CLP are summa-rized in Figure 4. As shown in Figure 4, the highest uptakewas in the heart (%ID/g: 0.12, p = 0.03), lung (%ID/g:0.49, p = 0.03), liver (%ID/g: 1.80), and spleen (%ID/g:1.63) in 30 min. The uptake decreased with time. The

Figure 1: Effect of pH on radiolabeling yield of 99mTc-DTPA-CLP.

Figure 2: Effect of stannous chloride on radiolabeling yield of99mTc-DTPA-CLP.

Figure 3: Serum stability results of 99mTc-DTPA-CLP.

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radiolabeled peptide was shown to be metabolized byhepatobiliary and urinary systems due to its low lipophilicity.Meanwhile, urine renal uptake decreased with time, whichis an indication of this situation. Donadio et al. demon-strated that liver in tumor-bearing rats showed a higheractivity of proenzyme (proMMP-9) compared with liversamples from untreated and control rats. In spleen,proMMP-9 and activated MMP-9 activities were both ele-vated in tumor-bearing rats, while control rats showed onlyminor activity with no differences between them (8). Caiet al. (9) reported that MMP-2 activity in lung cancers wassignificantly higher compared with normal lung using gela-tin zymography results. In the study by Kuhnast et al. (5)radioiodinated cyclic decapeptide HWGF had the highestuptake in the heart, liver, and kidney, but radioactivity wascleared quickly by the urinary tract in kidney. The datafrom the current study are consistent with previousreports.

The highest uptake was observed in the uterus (0.39,p = 0.03), ovary (0.15, p = 0.00), and breast (0.15,p = 0.03) at 30 min. MMP-2 and MMP-9 are overexpres-sed in different human epithelial cancers, such as breast,bladder, ovarian, and prostate cancer, and their levelsseem to be associated with malignancy and invasion (4).The high uptake in the uterus may be due to higherexpression of MMP-2 and MMP-9 in this tissue (10).Medina et al. investigated the effect of chemical modifica-tion and radiolabeling on CTT peptide activity. It wasobserved that the CTT decapeptide lost its gelatinaseinhibitory activity and could not be used as a tumor-target-ing agent in mice after direct labeling with 125I on its aro-matic residues. They also found that 99mTc-CTT was mosteffective when coated on the surface of liposomes, possi-bly indicating the suitable use of imaging a tumor siteusing a gamma camera in the real time (6).

Hanaoka et al. (2007) designed the gelatinase-selectiveinhibitor peptide, 111In-DTPA-CTT, which was observed toreduce the accumulation in non-target without impairinginhibitory activity. Hence, the accumulation of 111In-DTPA-

CTT reduced in the liver and kidney due to a highly hydro-philic and negatively charged In- chelate, In-DTPA wasattached to the amino group of the CTT peptide (2).

Sprague et al. (2006) described radiolabeling conjugatedDOTA-CTT with 64Cu for in vitro and in vivo studies.64Cu-DOTA-CTT had low tumor uptake due to poor in

vivo stability of the complex, suggesting that 64Cu-DOTA-CTT is not an optimal PET imaging agent (3). Ra-diolabeling of inhibitor peptide 68Ga-DOTA-TCTP-1 wasinvestigated as an imaging agent by Ujula et al. In theirstudy, uptake was observed in the heart, liver, kidney,and bladder. 68Ga-DOTA-TCTP-1 showed a high tumoruptake in melanoma rats by PET imaging (7). In ourstudy, 99mTc-DTPA was attached to an amino group ofthe N-terminal of cysteine of the CLP. It was observedthat the uptake of 99mTc-DTPA-CLP decreased in theorgans except for uterus, spleen, and breast comparedwith the uptake of 99mTc-CLP (11). Peptide-based radio-pharmaceuticals are useful for diagnostic applicationsdue to their high affinity and selectivity for target mole-cules (2,11,12). However, accumulation of peptide conju-gates is observed to decrease in non-target tissuessuch as the liver and kidney, thus resulting in good-quality imaging.

Gelatinases are potential targets for tumor imaging. Expres-sion of MMPs in normal tissue is generally lower comparedwith tumor tissue. Due to overexpression of MMP-9 intumor tissue, radiolabeled peptide, which is an inhibitorenzyme, is provided to construct imaging (2,5,7). In conclu-sion, accumulation of 99mTc-DTPA-CLP was observed insome organs (uterus, spleen, breast, etc.) related to MMPsin the normal rats. Our findings indicate that the matrix me-talloproteinase inhibitory peptide conjugate, DTPA-CLP,may contribute to construct new radiopharmaceuticals thatcan be used for the imaging of gelatinase activity in meta-static tumors overexpressing MMPs.

Conclusion

This study demonstrated that DTPA-CLP is labeled with99mTc with high yield. 99mTc-DTPA-CLP remained stable inserum. The radiolabeled peptide conjugate has a greateraffinity to those cells expressing matrix metalloproteinaseinhibitor as indicated by its accumulation in organs suchas uterus, ovary, liver, and breast. The results of this studyare significantly encouraging on further evaluation of theradiolabeled peptide conjugate as a possible gelatinaseexpression tumors imaging agent.

Acknowledgment

The authors gratefully acknowledge the financial supportreceived from the Department of Scientific Projects at EgeUniversity, Izmir, Turkey, and the Scientific and Technolog-

Figure 4: Biodistribution results of 99mTc-DTPA-CLP.

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ical Research Council of Turkey, Scientific Project(111S253).

Conflict of Interest

There is no conflict of interest in this manuscript.

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